PROJECT SUMMARY Objectives: Non-Hodgkin?s lymphomas (NHLs) are the 6th most common cancers in the US, of which 90% are B cells-type, and include 60% aggressive and 40% indolent. Although all types of NHL respond well to initial therapy, indolent NHLs are incurable: the disease relapses after the patient has achieved complete response (CR) to therapy. Also, despite efforts to develop effective therapies, many aggressive lymphomas recur and eventually 30% of these become incurable, highlighting the need to develop new therapies or to improve existing ones. In this proposal, we introduce a novel drug sensitization strategy for currently available anti-NHL drugs. The approach makes use of the sgRNA-SAM technology, a variant of the CRISPR tools, for effective, specific and sustained reactivation of the endogenous Ehd3 gene. The proposed approach is based on the fact that Ehd3 is preferentially down-regulated in a subpopulation of CD34+ cells isolated from NHL specimens and which show a reduced drug sensitivity. Therefore, our working hypothesis is that loss of Ehd3 expression in CD34+ cells is a driver event in the drug insensitivity of NHL cells. Consequently, restoring its expression will potentiate the cytotoxic effects of currently used drugs. Specific Aims: Aim1, will consist in a prospective study to assess Ehd3 expression in patient-derived NHL specimens. Here, we aim to identify specific subtypes of NHLs in which Ehd3 expression levels are predominantly decreased, and which could be more suitable for the proposed gene reactivation approach. In Aim2, we seek to perform in vitro development and test of the proposed sgRNA-SAM-directed Ehd3 transcriptional reactivation, in NHL established cell lines and patient-derived (PD) cells. We will also assess the power of the approach in potentiating the cells? sensitivity to the cytotoxic effects of 2CdA-R and CHOP-R drugs in vitro. In Aim3, we will perform in vivo proof-of-principle of sgRNA-SAM approach as a drug sensitization strategy, under the CHOP-R and 2CdA-R regimens, using animal xenografts of NHL cells.